Rat endopeptidase-24.18 α subunit is secreted into the culture medium as a zymogen when expressed by COS-1 cells

AbstractEndopeptidase-24.18 (EC 3.4.24.18, E-24.18) is an oligomeric Zn-ectoenzyme. The α and β submits have been cloned from both rat and mouse kidneys. The primary structure of these subunits revealed that they both contain the consensus Zn binding site and that they are members of the astacin family. Analysis of the hydropathy plot also suggested that they are anchored by a C-terminal hydrophobic domain. In order to verify the mode of anchoring of the rat E-24.18 α subunit and to test the functionality of the astacin-like domain in the α subunit when expressed alone, COS-1 cells were transfected with a cloned cDNA for rat α subunit. Despite the presence of its putative transmembrane domain, the α subunit was not anchored in the plasma membrane but rather secreted as a dimer into the culture medium. When the enzymatic activity of the secreted recombinant protein was tested in the azocasein degradation assay, the α subunit was found to be inactive. Activity could, however, be revealed after mild trypsin digestion. This activity was abolished by replacing the Glu-157 in the active site by Val. Taken together our results suggest that the α subunit of Endopeptidase-24.18 contains a latent astacin-like Zn metallopeptidase activity which could be secreted as a soluble enzyme by kidney and intestine

Applicability of GFP Microbial Whole Cell Biosensors to Bioreactor Operations : Mathematical Modeling and Related Experimental Tools

International audienc

Development of optical trajectography device for the lagangian study of turbulent flow inside a stirred tank used in pharmaceutical industry

Stirred tanks are devices that are widely used in various process steps of the pharmaceutical industry. The performance of these processes (animal cell culture, crystallization, flocculation …) strongly depend on the physico-chemical and hydrodynamic environment present locally in these tanks. To fully describe this local environment, an Eulerian - Lagrangian approach must be adopted in order to establish history of conditions met by a particle as a cell, a crystal or a floc. This approach implies to determine the trajectory followed by the particle. To this aim, the Chemical Laboratory of Liege University has developed a prototype of optical trajectography device. The objective of this paper is to present the device, developments that were necessary for its use and the results obtained. The device is composed of two cameras modeled by a pinhole model which record the position of a bead that has a size equal to 490 µm and that perfectly follows local flow structures. The measured trajectory has been validated by comparing average time velocity fields deduced from it to those measured, in the same operating conditions, by particle image velocimetry (P.I.V.).FRFC 2.4.579.09.

Investigation of the links between mass transfer conditions, dissolved hydrogen concentration and biohydrogen production by the pure strain Clostridium butyricum CWBI1009

Fermentative hydrogen production has often been described as inhibited by its own gas production. In this work, hydrogen production by Clostridium butyricum was investigated in batch Biochemical Hydrogen Potential (BHP) tests and in a 2.5L anaerobic sequenced batch reactor (AnSBR) under different operating conditions regarding liquid-to-gas mass transfer. Through the addition of both stirring up to 400rpm and nitrogen sparging, the yields were enhanced from 1.6 to 3.1molH2molglucose -1 and the maximum hydrogen production rates from 140 to 278mLh-1. These original results were achieved with a pure Clostridium strain. They showed that hydrogen production was improved by a higher liquid-to-gas hydrogen transfer resulting in a lower dissolved hydrogen concentration in the culture medium and therefore in a lower bacterial inhibition. In addition, biohydrogen partitioning between the gas and the liquid phase did not conform to Henry's Law due to critical supersaturation phenomena up to seven-fold higher than the equilibrium conditions. Therefore, dissolved hydrogen concentration should be systematically measured instead of the headspace hydrogen partial pressure. A model was proposed to correlate H2 production yield and rate by the pure C. butyricum strain CWBI1009 with mass transfer coefficient KLa.Etude de la production d'hydrogène par les bactéries anaérobies chimiotrophes (dark-fermentation

Caractérisation des perturbations extracellulaires induites au sein des réacteurs scale-down par la superposition d'une approche expérimentale et d'une approche numérique

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